Regulation of all cellular processes requires dynamic regulation of protein phosphorylation. We have developed an unbiased system to globally quantify the phosphorylation index for substrates of a specific kinase by independently quantifying phosphorylated and total substrate molecules in a reverse in-gel kinase assay. Non-phosphorylated substrate molecules are first quantified in the presence and absence of a specific stimulus. Total substrate molecules are then measured after complete chemical de-phosphorylation, and a ratio of phosphorylated to total substrate is derived. To demonstrate the utility of this approach, we profiled and quantified changes in phosphorylation index for Protein Kinase CK2 substrates that respond to a small-molecule inhibitor. A broad range of inhibitor-induced changes in phosphorylation was observed in cultured cells. Differences among substrates in the kinetics of phosphorylation change were also revealed. Comparison of CK2 inhibitor-induced changes in phosphorylation in cultured cells and in mouse peripheral blood lymphocytes in vivo revealed distinct kinetic and depth-of-response profiles. This technology provides a new approach to facilitate functional analyses of kinase-specific phosphorylation events. This strategy can be used to dissect the role of phosphorylation in cellular events, to facilitate kinase inhibitor target validation studies, and to inform in vivo analyses of kinase inhibitor drug efficacy.